Lettuce variety inferno

ABSTRACT

The present invention provides novel lettuce cultivar Inferno and plant parts, seed, and tissue culture therefrom. The invention also provides methods for producing a lettuce plant by crossing the lettuce plants of the invention with themselves or another lettuce plant. The invention also provides lettuce plants produced from such a crossing as well as plant parts, seed, and tissue culture therefrom.

FIELD OF THE INVENTION

This invention is in the field of lettuce plants, in particular, theinvention relates to novel romaine lettuce plants characterized by darkgreen color, slow bolting and disease resistance.

BACKGROUND OF THE INVENTION

The present invention relates to a romaine lettuce (Lactuca sativa L.)variety designated Inferno.

Practically speaking, all cultivated forms of lettuce belong to thehighly polymorphic species Lactuca sativa that is grown for its ediblehead and leaves. Lactuca sativa is in the Cichoreae tribe of theAsteraceae (Compositae) family. Lettuce is related to chicory,sunflower, aster, dandelion, artichoke, and chrysanthemum. Sativa is oneof about 300 species in the genus Lactuca. There are seven differentmorphological types of lettuce. The crisphead group includes the icebergand batavian types. Iceberg lettuce has a large, firm head with a crisptexture and a white or creamy yellow interior. The batavian lettucepredates the iceberg type and has a smaller and less firm head. Thebutterhead group has a small, soft head with an almost oily texture. Theromaine, also known as cos lettuce, has elongated upright leaves forminga loose, loaf-shaped head and the outer leaves are usually dark green.Leaf lettuce comes in many varieties, none of which form a head, andinclude the green oak leaf variety. Latin lettuce looks like a crossbetween romaine and butterhead. Stem lettuce has long, narrow leaves andthick, edible stems. Oilseed lettuce is a type grown for its large seedsthat are pressed to obtain oil. Latin lettuce, stem lettuce, and oilseedlettuce are seldom seen in the United States.

Presently, there are over one thousand known lettuce cultivars. As acrop, lettuce is grown commercially wherever environmental conditionspermit the production of an economically viable yield.

Lettuce in general, and leaf lettuce in particular, is an important andvaluable vegetable crop. Thus, there is an ongoing need for improvedlettuce varieties.

SUMMARY OF THE INVENTION

According to the invention, there is provided a novel lettuce cultivardesignated Inferno, also known as LS10884, having desirablecharacteristics including dark green color, slow bolting and diseaseresistances to Tomato Bushy Stunt virus [TBSV], Big Vein virus and mostCalifornia races of downy mildew. Thus, the invention also encompassesthe seeds of lettuce cultivar Inferno, the plants of lettuce cultivarInferno, plant parts of the lettuce cultivar Inferno (including leaves,seed, gametes), methods of producing seed from lettuce cultivar Inferno,and method for producing a lettuce plant by crossing the lettucecultivar Inferno with itself or another lettuce plant, methods forproducing a lettuce plant containing in its genetic material one or moretransgenes, and the transgenic lettuce plants produced by that method.The invention also relates to methods for producing other lettuce plantsderived from lettuce cultivar Inferno and to lettuce plants, partsthereof and seed derived by the use of those methods. The presentinvention further relates to hybrid lettuce seeds and plants (and partsthereof including leaves) produced by crossing lettuce cultivar Infernowith another lettuce plant.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of lettuce cultivar Inferno. In embodiments, thetissue culture is capable of regenerating plants having all oressentially all of the physiological and morphological characteristicsof the foregoing lettuce plant and/or of regenerating plants having thesame or substantially the same genotype as the foregoing lettuce plant.In exemplary embodiments, the regenerable cells in such tissue culturesare meristematic cells, cotyledons, hypocotyl, leaves, pollen, embryos,roots, root tips, anthers, pistils, ovules, shoots, stems, petiole,pith, flowers, capsules and/or seeds as well as callus and/orprotoplasts derived from any of the foregoing. Still further, thepresent invention provides lettuce plants regenerated from the tissuecultures of the invention.

As a further aspect, the invention provides a method of producinglettuce seed, the method comprising crossing a plant of lettuce cultivarInferno with itself or a second lettuce plant. Optionally, the methodfurther comprises collecting the seed.

Another aspect of the invention provides methods for producing hybridsand other lettuce plants derived from lettuce cultivar Inferno. Lettuceplants derived by the use of those methods are also part of theinvention as well as plant parts, seed, gametes and tissue culture fromsuch hybrid or derived lettuce plants.

In representative embodiments, a lettuce plant derived from lettucecultivar Inferno comprises cells comprising at least one set ofchromosomes derived from lettuce cultivar Inferno. In embodiments, alettuce plant or population of lettuce plants derived from lettucecultivar Inferno comprises, on average, at least 6.25%, 12.5%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%,98% or 99% of its alleles from lettuce cultivar Inferno. In embodiments,the lettuce plant derived from lettuce cultivar Inferno is one, two,three, four, five or more breeding crosses removed from lettuce cultivarInferno.

In embodiments, a hybrid or derived plant from lettuce cultivar Infernocomprises a desired added trait(s). In representative embodiments, alettuce plant derived from lettuce cultivar Inferno comprises all of themorphological and physiological characteristics of lettuce cultivarInferno (e.g., as described in Table 1). In embodiments, the lettuceplant derived from lettuce cultivar Inferno comprises essentially all ofthe morphological and physiological characteristics of lettuce cultivarInferno (e.g., as described in Table 1), with the addition of a desiredadded trait(s).

The invention also relates to methods for producing a lettuce plantcomprising in its genetic material one or more transgenes and to thetransgenic lettuce plant produced by those methods (and progeny lettuceplants comprising the transgene). Also provided are plant parts, seedand tissue culture from such transgenic lettuce plants, optionallywherein one or more cells in the plant part, seed, or tissue culturecomprises the transgene. The transgene can be introduced via planttransformation and/or breeding techniques.

In another aspect, the present invention provides for single geneconverted plants of lettuce cultivar Inferno. Plant parts, seed, andtissue culture from such single gene converted plants are alsocontemplated by the present invention. The single transferred gene maybe a dominant or recessive allele. In representative embodiments, thesingle transferred gene confers such traits as male sterility, herbicideresistance, pest resistance (e.g., insect and/or nematode resistance),modified fatty acid metabolism, modified carbohydrate metabolism,disease resistance (e.g., for bacterial, fungal and/or viral disease),male fertility, enhanced nutritional quality, improved appearance (e.g.,color), improved salt tolerance, industrial usage, or any combinationthereof. The single gene may be a naturally occurring lettuce gene or atransgene introduced into lettuce through genetic engineeringtechniques.

The invention further provides methods for developing lettuce plants ina lettuce plant breeding program using plant breeding techniquesincluding, for example, recurrent selection, backcrossing, pedigreebreeding, double haploid techniques, restriction fragment lengthpolymorphism enhanced selection, genetic marker enhanced selectionand/or transformation. Seeds, lettuce plants, and parts thereof,produced by such breeding methods are also part of the invention.

The invention also provides methods of multiplication or propagation oflettuce plants of the invention, which can be accomplished using anymethod known in the art, for example, via vegetative propagation and/orseed.

The invention further provides a method of producing food or feedcomprising (a) obtaining a lettuce plant of the invention, optionallywherein the plant has been cultivated to maturity, and (b) collecting atleast one lettuce plant or part thereof (e.g., leaves) from the plant.

Additional aspects of the invention include harvested products andprocessed products from the lettuce plants of the invention. A harvestedproduct can be a whole plant or any plant part, as described herein.Thus, in some embodiments, a non-limiting example of a harvested productincludes a seed, a leaf and/or a stem.

In representative embodiments, a processed product includes, but is notlimited to: cut, sliced, ground, pureed, dried, canned, jarred, washed,packaged, frozen and/or heated leaves and/or seeds of the lettuce plantsof the invention, or any other part thereof. In embodiments, a processedproduct includes a sugar or other carbohydrate, fiber, protein and/oraromatic compound that is extracted, purified or isolated from a lettuceplant of the invention. In embodiments, the processed product includeswashed and packaged leaves (or parts thereof) of the invention.

The seed of the invention can optionally be provided as an essentiallyhomogenous population of seed of a single plant or cultivar. Essentiallyhomogenous populations of seed are generally free from substantialnumbers of other seed, e.g., at least about 90%, 95%, 96%, 97%, 98% or99% pure.

In representative embodiments, the invention provides a seed of lettucecultivar Inferno.

As a further aspect, the invention provides a plant of lettuce cultivarInferno.

As an additional aspect, the invention provides a lettuce plant, or apart thereof, having all or essentially all of the physiological andmorphological characteristics of a plant of lettuce cultivar Inferno.

As another aspect, the invention provides leaves and/or seed of thelettuce plants of the invention and a processed product from the leavesand/or seed of the inventive lettuce plants.

As still another aspect, the invention provides a method of producinglettuce seed, the method comprising crossing a lettuce plant of theinvention with itself or a second lettuce plant. The invention alsoprovides seed produced by this method and plants produced by growing theseed.

As yet a further aspect, the invention provides a method for producing aseed of a lettuce plant derived from lettuce cultivar Inferno, themethod comprising: (a) crossing a lettuce plant of lettuce cultivarInferno with a second lettuce plant; and (b) allowing seed of a lettuceplant derived from lettuce cultivar Inferno to form. In embodiments, themethod further comprises: (c) growing a plant from the seed derived fromlettuce cultivar Inferno of step (b); (d) selfing the plant grown fromthe lettuce seed derived from lettuce cultivar Inferno or crossing it toa second lettuce plant to form additional lettuce seed derived fromlettuce cultivar Inferno, and (e) repeating steps (c) and (d) 0 or moretimes to generate further derived lettuce seed. Optionally, the methodcomprises: (e) repeating steps (c) and (d) one or more times (e.g., oneto three, one to five, one to six, one to seven, one to ten, three tofive, three to six, three to seven, three to eight or three to tentimes) to generate further derived lettuce plants. As another option,the method can comprise collecting the seed. The invention also providesseed produced by these methods and plants produced by growing the seed.

As another aspect, the invention provides a method of producing lettuceleaves, the method comprising: (a) obtaining a plant of lettuce cultivarInferno, optionally wherein the plant has been cultivated to maturity;and (b) collecting leaves from the plant. The invention also providesthe leaves produced by this method.

Still further, as another aspect, the invention provides a method ofvegetatively propagating a plant of lettuce cultivar Inferno. In anon-limiting example, the method comprises: (a) collecting tissuecapable of being propagated from a plant of lettuce cultivar Inferno;(b) cultivating the tissue to obtain proliferated shoots; and (c)rooting the proliferated shoots to obtain rooted plantlets. Optionally,the invention further comprises growing plants from the rootedplantlets. The invention also encompasses the plantlets and plantsproduced by these methods.

As an additional aspect, the invention provides a method of introducinga desired added trait into lettuce cultivar Inferno, the methodcomprising: (a) crossing a first plant of lettuce cultivar Inferno witha second lettuce plant that comprises a desired trait to produce F₁progeny; (b) selecting an F₁ progeny that comprises the desired trait;(c) crossing the selected F₁ progeny with lettuce cultivar Inferno toproduce backcross progeny; and (d) selecting backcross progenycomprising the desired trait to produce a plant derived from lettucecultivar Inferno comprising a desired trait. In embodiments, theselected progeny has a dark green color. In embodiments, the selectedprogeny has slow bolting. In embodiments, the selected progeny hasresistance to TBSV and/or downy mildew (e.g., most California races ofdowny mildew). In embodiments, the selected progeny is tolerant to tipburn, heat stress and/or cold stress. In embodiments, the selectedprogeny comprises all or essentially all the morphological andphysiological characteristics of the first plant of lettuce cultivarInferno. Optionally, the method further comprises: (e) repeating steps(c) and (d) one or more times in succession (e.g., one to three, one tofive, one to six, one to seven, one to ten, three to five, three to six,three to seven, three to eight or three to ten times) to produce a plantderived from lettuce cultivar Inferno comprising the desired trait.

In representative embodiments, the invention also provides a method ofproducing a plant of lettuce cultivar Inferno comprising a desired addedtrait, the method comprising introducing a transgene conferring thedesired trait into a plant of lettuce cultivar Inferno. The transgenecan be introduced by transformation methods (e.g., genetic engineering)or breeding techniques. In embodiments, the plant comprising thetransgene has very a dark green color. In embodiments, the plantcomprising the transgene has slow bolting. In embodiments, the plantcomprising the transgene is resistant to TBSV and/or downy mildew (e.g.,most California races of downy mildew). In embodiments, the plantcomprising the transgene is tolerant to tip burn, heat stress and/orcold stress. In embodiments, the plant comprising the transgenecomprises all or essentially all of the morphological and physiologicalcharacteristics of lettuce cultivar Inferno.

The invention also provides lettuce plants produced by the methods ofthe invention, wherein the lettuce plant has the desired added trait aswell as seed from such lettuce plants.

According to the foregoing methods, the desired added trait can be anysuitable trait known in the art including, for example, male sterility,male fertility, herbicide resistance, insect or pest (e.g., insectand/or nematode) resistance, modified fatty acid metabolism, modifiedcarbohydrate metabolism, disease resistance (e.g., for bacterial, fungaland/or viral disease), enhanced nutritional quality, increasedsweetness, increased flavor, improved ripening control, improved salttolerance, industrial usage, or any combination thereof.

In representative embodiments, a transgene conferring herbicideresistance confers resistance to glyphosate, sulfonylurea,imidazolinone, dicamba, glufosinate, phenoxy proprionic acid,L-phosphinothricin, cyclohexone, cyclohexanedione, triazine,benzonitrile, or any combination thereof.

In representative embodiments, a transgene conferring pest resistance(e.g., insect and/or nematode resistance) encodes a Bacillusthuringiensis endotoxin.

In representative embodiments, transgenic plants, transformed plants(e.g., using genetic engineering techniques), single gene convertedplants, hybrid plants and lettuce plants derived from lettuce cultivarInferno are characterized by dark green color and/or slow bolting and/orresistance to TBSV and/or downy mildew and/or tolerance to tip burn,heat stress and/or cold stress. In representative embodiments,transgenic plants, transformed plants, hybrid plants and lettuce plantsderived from lettuce cultivar Inferno have at least 3, 4, 5, 6, 7, 8, 9,10 or more of the morphological and physiological characteristics oflettuce cultivar Inferno (e.g., as described in Table 1), or even all ofthe morphological and physiological characteristics of lettuce cultivarInferno, so that said plants are not significantly different for saidtraits than lettuce cultivar Inferno, as determined at the 5%significance level when grown in the same environmental conditions;optionally, with the presence of one or more desired additional traits(e.g., male sterility, disease resistance, pest or insect resistance,herbicide resistance, and the like).

The invention also encompasses plant parts, plant material, pollen,ovules, leaves, fruit and seed from the lettuce plants of the invention.Also provided is a tissue culture of regenerable cells from the lettuceplants of the invention, where optionally, the regenerable cells are:(a) embryos, meristem, leaves, pollen, cotyledons, hypocotyls, roots,root tips, anthers, flowers, pistils, ovules, seed, shoots, stems,stalks, petioles, pith and/or capsules; or (b) callus or protoplastsderived from the cells of (a). Further provided are lettuce plantsregenerated from a tissue culture of the invention.

In addition to the exemplary aspects and embodiments described above,the invention is described in more detail in the description of theinvention set forth below.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the development of a novelromaine lettuce having desirable characteristics including dark greencolor, slow bolting and disease resistances to TBSV and most Californiaraces of downy mildew.

It should be appreciated that the invention can be embodied in differentforms and should not be construed as limited to the embodiments setforth herein. Rather, these embodiments are provided so that thisdisclosure will be thorough and complete, and will fully convey thescope of the invention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

Unless the context indicates otherwise, it is specifically intended thatthe various features and embodiments of the invention described hereincan be used in any combination.

Moreover, the present invention also contemplates that in someembodiments of the invention, any feature or combination of features setforth herein can be excluded or omitted. To illustrate, if thespecification states that a composition comprises components A, B and C,it is specifically intended that any of A, B or C, or a combinationthereof, can be omitted and disclaimed singularly or in any combination.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

DEFINITIONS

In the description and tables that follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

As used in the description of the invention and the appended claims, thesingular forms “a,” “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

As used herein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

The term “about,” as used herein when referring to a measurable valuesuch as a dosage or time period and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of thespecified amount.

The term “comprise,” “comprises” and “comprising” as used herein,specify the presence of the stated features, integers, steps,operations, elements, and/or components, but do not preclude thepresence or addition of one or more other features, integers, steps,operations, elements, components, and/or groups thereof.

As used herein, the transitional phrase “consisting essentially of”means that the scope of a claim is to be interpreted to encompass thespecified materials or steps recited in the claim “and those that do notmaterially affect the basic and novel characteristic(s)” of the claimedinvention. See, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463(CCPA 1976) (emphasis in the original); see also MPEP §2111.03. Thus,the term “consisting essentially of” when used in a claim or thedescription of this invention is not intended to be interpreted to beequivalent to “comprising.”

“Allele”. An allele is any of one or more alternative forms of a gene,all of which relate to a trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

“Backcrossing”. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

“Big Vein virus”. Big vein is a disease of lettuce caused by LettuceMirafiori Big Vein Virus which is transmitted by the fungus Olpidiumvirulentus, with vein clearing and leaf shrinkage resulting in plants ofpoor quality and reduced marketable value.

“Bolting”. The premature development of a flowering stalk, andsubsequent seed, before a plant produces a food crop. Bolting istypically caused by late planting when temperatures are low enough tocause vernalization of the plants.

“Bremia lactucae”. An Oomycete that causes downy mildew in lettuce incooler growing regions.

“Core length”. Length of the internal lettuce stem measured from thebase of the cut and trimmed head to the tip of the stem.

“Corky root”. A disease caused by the bacterium Sphingomonassuberifaciens, which causes the entire taproot to become brown, severelycracked, and non-functional.

“Cotyledon”. One of the first leaves of the embryo of a seed plant;typically one or more in monocotyledons, two in dicotyledons, and two ormore in gymnosperms.

“Double haploid line”. A stable inbred line achieved by doubling thechromosomes of a haploid line, e.g., from anther culture. For example,some pollen grains (haploid) cultivated under specific conditionsdevelop plantlets containing 1n chromosomes. The chromosomes in theseplantlets are then induced to “double” (e.g., using chemical means)resulting in cells containing 2n chromosomes. The progeny of theseplantlets are termed “double haploid” and are essentially notsegregating any more (e.g., are stable). The term “double haploid” isused interchangeably herein with “dihaploid.”

“Essentially all the physiological and morphological characteristics”. Aplant having “essentially all the physiological and morphologicalcharacteristics” means a plant having the physiological andmorphological characteristics of the recurrent parent, except for thecharacteristics derived from the converted gene(s).

“First water date”. The date the seed first receives adequate moistureto germinate. This can and often does equal the planting date.

“Gene”. As used herein, “gene” refers to a segment of nucleic acidcomprising an open reading frame. A gene can be introduced into a genomeof a species, whether from a different species or from the same species,using transformation or various breeding methods.

“Head diameter”. Diameter of the cut and trimmed head, slicedvertically, and measured at the widest point perpendicular to the stem.

“Head height”. Height of the cut and trimmed head, sliced vertically,and measured from the base of the cut stem to the cap leaf.

“Head weight”. Weight of saleable lettuce head, cut and trimmed tomarket specifications.

“Inbred line”: As used herein, the phrase “inbred line” refers to agenetically homozygous or nearly homozygous population. An inbred line,for example, can be derived through several cycles of sib crossingand/or selfing and/or via double haploid production. In someembodiments, inbred lines breed true for one or more traits of interest.An “inbred plant” or “inbred progeny” is an individual sampled from aninbred line.

“Lettuce Mosaic virus”. A disease that can cause a stunted, deformed, ormottled pattern in young lettuce and yellow, twisted, and deformedleaves in older lettuce.

“Maturity date”. Maturity refers to the stage when the plants are offull size and/or optimum weight and/or in marketable form to be ofcommercial or economic value.

“Nasonovia ribisnigri”. A lettuce aphid that colonizes the innermostleaves of the lettuce plant, contaminating areas that cannot be treatedeasily with insecticides.

“Plant.” As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants, such as leaves, pollen, embryos,cotyledons, hypocotyl, roots, root tips, anthers, pistils, flowers,ovules, seeds, fruit, stems, and the like.

“Plant material”. The terms “plant material” and “material obtainablefrom a plant” are used interchangeably herein and refer to any plantmaterial obtainable from a plant including without limitation, leaves,stems, roots, flowers or flower parts, fruits, pollen, ovules, zygotes,seeds, cuttings, cell or tissue cultures, or any other part or productof the plant.

“Plant part”. As used herein, a “plant part” includes any part, organ,tissue or cell of a plant including without limitation an embryo,meristem, leaf, pollen, cotyledon, hypocotyl, root, root tip, anther,flower, flower bud, pistil, ovule, seed, shoot, stem, stalk, petiole,pith, capsule, a scion, a rootstock and/or a fruit including callus andprotoplasts derived from any of the foregoing.

“Quantitative Trait Loci”. Quantitative Trait Loci (QTL) refers togenetic loci that control to some degree, numerically representabletraits that are usually continuously distributed.

“Ratio of head height/diameter”. Head height divided by the headdiameter is an indication of the head shape; <1 is flattened, 1=round,and >1 is pointed.

“Regeneration”. Regeneration refers to the development of a plant fromtissue culture.

“Resistance”. As used herein the terms “resistance” and “tolerance” (andgrammatical variations thereof) are used interchangeably to describeplants that show reduced or essentially no symptoms to a specific biotic(e.g., a pest, pathogen or disease) or abiotic (e.g., exogenous orenvironmental, including herbicides) factor or stressor. In someembodiments, “resistant” or “tolerant” plants show some symptoms but arestill able to produce marketable product with an acceptable yield, e.g.,the yield may still be reduced and/or the plants may be stunted ascompared with the yield or growth in the absence of the biotic and/orabiotic factor or stressor. Those skilled in the art will appreciatethat the degree of resistance or tolerance may be assessed with respectto a plurality or even an entire field of plants. A lettuce plant may beconsidered “resistant” or “tolerant” if resistance/tolerance is observedover a plurality of plants (e.g., an average), even if particularindividual plants may be susceptible to the biotic or abiotic factor orstressor.

“RHS”. RHS refers to the Royal Horticultural Society of England whichpublishes an official botanical color chart quantitatively identifyingcolors according to a defined numbering system. The chart may bepurchased from Royal Horticulture Society Enterprise Ltd., RHS Garden;Wisley, Woking; Surrey GU236QB, UK.

“Single gene converted”. A single gene converted or conversion plantrefers to a plant that is developed by plant breeding techniques (e.g.,backcrossing) or via genetic engineering wherein essentially all of thedesired morphological and physiological characteristics of a line arerecovered in addition to the single gene transferred into the line viathe plant breeding technique or via genetic engineering.

“Substantially equivalent characteristic”. A characteristic that, whencompared, does not show a statistically significant difference (e.g.,p=0.05) from the mean.

“Tip burn”. Means a browning of the edges or tips of lettuce leaves thatis a physiological response to a lack of calcium.

“Tomato Bushy Stunt”. Also called “lettuce necrotic stunt”. A diseasethat causes stunting of growth and leaf mottling.

“Transgene”. A nucleic acid of interest that can be introduced into thegenome of a plant by genetic engineering techniques (e.g.,transformation) or breeding. The transgene can be from the same or adifferent species. If from the same species, the transgene can be anadditional copy of a native coding sequence or can present the nativesequence in a form or context (e.g., different genomic location and/orin operable association with exogenous regulatory elements such as apromoter) than is found in the native state. The transgene can comprisean open reading frame encoding a polypeptide or can encode a functionalnon-translated RNA (e.g., RNAi).

Botanical Description of the Lettuce Cultivar Inferno.

Lettuce Inferno is a dark green romaine lettuce variety suitable forfull size production in the coastal areas of California in the Spring,Summer and Fall harvesting seasons, and the Southwest deserts ofCalifornia and Arizona in the late Fall and early Spring harvestingseasons. Lettuce variety Inferno resulted from a three-way cross oflettuce varieties and subsequent numerous generations of individualplant selections chosen for their dark green color, slow bolting anddisease resistances.

Lettuce Inferno has shown uniformity and stability for these traits,within the limits of environmental influence for the traits. It has beenself-pollinated a sufficient number of generations with carefulattention to uniformity of plant type. The variety has been increasedwith continued observation for uniformity. No variant traits have beenobserved or are expected in cultivar Inferno.

TABLE 1 Variety Description Information. Characteristics. Lettucecultivar Inferno is characterized by dark green color, slow bolting, andresistances to Tomato Bushy Stunt virus (TBSV) and most California racesof downy mildew. Plant Type: Green romaine Seed a. Color: Black b. Lightdormancy: Light not required c. Heat dormancy: Susceptible Cotyledon toFourth Leaf Stage a. Shape of cotyledons: Broad b. Undulation: Flat c.Anthocyanin distribution: Absent d. Rolling: Absent e. Cupping: Uncappedf. Reflexing: None Mature Leaves a. Margin Incision depth:Absent/Shallow b. Margin Indentation: Entire c. Margin Undulation of theapical margin: Absent/Slight d. Green color: Very Dark Green e.Anthocyanin Distribution: Absent f. Glossiness: Glossy g. Blistering:Moderate h. Trichomes: Absent i. Leaf thickness: Thick Plant at MarketStage a. Head shape: Slight V Shaped b. Head size class: Medium c. Headweight (g): 827.08 d. Head firmness: Loose Core a. Diameter at base ofhead (cm): 3.43 b. Core height from base of head to apex (cm): 7.86Maturity (days) a. Summer: 65 b. Winter: 115 Adaptation a. PrimaryRegions of Adaptation (tested and proven adapted) b. Southwest(California, Arizona desert): Yes c. West Coast: Yes i. Spring area:Salinas, Yuma, Imperial, San Joaquin ii. Summer area: Salinas, SantaMaria, San Benito iii. Fall area: Yuma, Imperial, Salinas, iv. Winterarea: Yuma, Imperial, Coachella d. Soil Type: Both of Mineral andOrganic Disease and Stress Reactions Virus a. TBSV: Highly resistant b.Big Vein: Intermediate c. Lettuce Mosaic: Not tested d. Cucumber Mosaic:Not tested e. Broad Bean Wilt: Not tested f. Turnip Mosaic: Not testedg. Best Western Yellows: Not tested h. Lettuce Infectious Yellows: Nottested Fungal/Bacterial a. Corky Root Rot (Pythium Root Rot): Not testedb. Downy Mildew: Highly resistant c. Powdery Mildew: Not tested d.Sclerotinia Rot: Not tested e. Bacterial Soft Rot (Pseudomonas sp. &others): Not tested f. Botrytis (Gray Mold): Not tested Insects a.Cabbage Loopers: Not tested b. Root Aphids: Not tested c. Green PeachAphid: Not tested Physiological/Stress a. Tip burn: Highly Tolerant b.Heat: Intermediate c. Drought: Not tested d. Cold: Tolerant e. Salt: Nottested f. Brown Rib: Not tested Post-Harvest a. Pink Rib: Not tested b.Russet Spotting: Not tested c. Rusty Brown Discoloration: Not tested d.Internal Rib Necrosis (Blackheart, Gray Rib, Gray Streak): Not tested e.Brown Stain: Not tested

TABLE 2 The length of cotyledon leaf measured in mm at 20 days oldseedlings Cotyledon Inferno length (mm) (Inferno) Green Thunder Del Sol19 22 16 16 19 16 22 18 18 19 19 18 17 19 19 12 16 17 17 23 17 15 19 1717 23 16 17 22 16 19 16 18 17 21 17 13 21 17 18 22 15 18 22 19 19 18 2017 22 14 20 20 18 19 18 19 18 22 16 Variety Mean N Duncan GroupingInferno 17.45 20 B Green Thunder 20.10 20 A Del Sol 17.15 20 B ANOVASource of Variation Df SS MS F P-value Variety 2 105.433 52.717 12.88<.0001 Anova shows a significant difference in the length of cotyledonleaf measured in mm at 20 days old seedlings, and the average cotyledonlength is 17.45, 20.10 and 17.15, respectively.

TABLE 3 The width of cotyledon leaf measured in mm at 20 days oldseedlings Cotyledon Inferno Width (mm) (Inferno) Green Thunder Del Sol 99 8 9 9 8 10 8 8 11 9 9 10 9 8 9 9 8 9 9 8 8 9 7 9 9 8 10 8 7 9 8 8 9 97 9 9 9 8 9 7 9 8 8 8 9 8 8 9 7 8 9 8 10 9 9 10 9 8 Variety Mean NDuncan Grouping Inferno 9.10 20 A Green Thunder 8.80 20 A Del Sol 7.9020 B ANOVA Source of Variation df SS MS F P-value Variety 2 15.600 7.80017.93 <.0001 Anova shows a significant difference in the width ofcotyledon leaf measured in mm at 20 days old seedlings, and the averagecotyledon width (cm) is 9.10, 8.80 and 7.90, respectively.

TABLE 4 Cotyledon Index calculated by dividing the cotyledon leaf lengthby the cotyledon leaf width Inferno Cotyledon Index (Inferno) GreenThunder Del Sol 2.1 2.4 2.0 1.8 2.1 2.0 2.3 2.3 2.3 1.7 2.1 2.0 1.7 2.12.4 1.3 1.8 2.1 1.9 2.6 2.1 1.9 2.1 2.4 1.9 2.6 2.0 1.7 2.8 2.3 2.1 2.02.3 1.9 2.3 2.4 1.4 2.3 1.9 2.3 2.4 2.1 2.0 2.8 2.4 2.4 2.0 2.5 2.1 2.42.0 2.5 2.2 2.3 1.9 2.0 2.1 1.8 2.4 2.0 Variety Mean N Duncan GroupingInferno 1.94 20 B Green Thunder 2.29 20 A Del Sol 2.18 20 A ANOVA Sourceof Variation Df SS MS F P-value Variety 2 1.290 0.645 9.57 0.0003 Anovashows a significant difference in the cotyledon leaf index calculated bydividing the cotyledon leaf length by the cotyledon leaf width measuredin mm at 20 days old seedlings, and the average cotyledon leaf index is1.94, 2.29 and 2.18, respectively.

TABLE 5 The length of the 4th true leaf measured in cm at a 20 days oldseedling Inferno Green 4th Leaf Length (cm) (Inferno) Thunder Del Sol2.9 4.9 5.2 3.4 3.1 4.7 3.4 4.0 5.5 3.9 3.2 4.3 3.5 3.2 5.8 3.0 4.4 4.53.2 3.9 4.5 2.7 4.2 5.3 3.3 4.0 5.0 3.9 4.2 4.2 3.0 4.3 4.9 3.6 4.0 6.02.6 4.3 5.9 3.0 3.9 4.4 3.2 3.4 3.8 4.2 4.1 5.3 3.8 4.2 5.6 4.9 3.1 4.73.2 3.2 5.1 . 3.2 . Variety Mean N Duncan Grouping Inferno 3.41 19 CGreen Thunder 3.84 20 B Del Sol 4.98 19 A ANOVA Source of Variation dfSS MS F P-value Variety 2 25.333 12.667 39.09 <.0001 Anova shows asignificant difference in the length of the 4th true leaf measured in cmat a 20 days old seedling, and the average 4th leaf length (cm) is 3.41,3.84 and 4.98, respectively.

TABLE 6 The width of the 4th true leaf measured in mm at a 20 days oldseedling Inferno Green 4th Leaf Width (cm) (Inferno) Thunder Del Sol 1.72.2 2.1 2.0 1.6 1.8 1.7 1.9 2.0 2.0 1.8 1.8 2.0 2.2 2.2 1.2 2.0 1.8 1.72.1 1.7 1.4 2.4 2.0 2.0 2.1 2.1 1.9 2.1 1.8 1.6 1.9 2.1 2.0 1.9 2.2 1.92.1 2.5 1.6 1.9 1.8 1.7 1.7 1.6 2.2 2.1 2.4 2.2 2.0 2.0 2.6 1.4 1.9 1.71.5 2.0 . 1.7 . Variety Mean N Duncan Grouping Inferno 1.85 19 A GreenThunder 1.93 20 A Del Sol 1.99 19 A ANOVA Source of Variation df SS MS FP-value Variety 2 0.194 0.097 1.34 0.2715 Anova shows no significantdifference in the width of the 4th true leaf measured in cm at a 20 daysold seedling.

TABLE 7 4th Leaf Index calculated by dividing the 4th leaf length by the4th leaf width Inferno Green 4th Leaf Index (Inferno) Thunder Del Sol1.7 2.2 2.5 1.7 1.9 2.6 2.0 2.1 2.8 2.0 1.8 2.4 1.8 1.5 2.6 2.5 2.2 2.51.9 1.9 2.6 1.9 1.8 2.7 1.7 1.9 2.4 2.1 2.0 2.3 1.9 2.3 2.3 1.8 2.1 2.71.4 2.0 2.4 1.9 2.1 2.4 1.9 2.0 2.4 1.9 2.0 2.2 1.7 2.1 2.8 1.9 2.2 2.51.9 2.1 2.6 . 1.9 . Variety Mean N Duncan Grouping Inferno 1.87 19 CGreen Thunder 2.01 20 B Del Sol 2.51 19 A ANOVA Source of Variation dfSS MS F P-value Variety 2 4.312 2.156 59.76 <.0001 Anova shows asignificant difference in 4th leaf index at a 20 days old seedling, andthe average 4th leaf index is 1.87, 2.01 and 2.51, respectively.

TABLE 8 Plant Weight(g) at Harvest Maturity Trial location Loc. 1 Loc. 2Loc. 3 Loc. 4 Inferno 859 1040 593 905 (Inferno) 763 932 626 931 8531395 614 844 890 1013 595 797 850 854 656 729 780 912 527 624 765 1021691 982 855 1012 770 732 905 900 628 803 780 1184 545 928 Green Thunder909 1428 827 1509 905 715 686 1235 661 826 648 830 900 822 704 920 850912 937 1381 850 963 787 918 860 1326 737 1379 780 1067 922 1093 9001070 702 1070 950 682 816 1041 Del Sol 995 1348 868 1292 853 1411 8011197 1085 1132 757 1387 1000 1357 727 1494 1100 963 711 988 1050 760 8521052 985 1422 785 1023 900 1081 938 1110 895 1440 755 1004 800 1281 8651336 Plant weight (g) SUMMARY Duncan Variety Mean N Grouping Inferno827.08 40 C Green Thunder 937.95 40 B Del Sol 1045.00 40 A ANOVA Sourceof Variation df SS MS F P-value Variety 2 949923.650 474961.825 20.68<.0001 Location 3 2266833.358 755611.119 32.89 <.0001 Interaction 6427902.417 71317.069 3.10 0.0076 Anova shows significant differences inplant weight in varieties, locations and significant interactionsbetween Variety and location. The average plant weight (g) of Inferno,Green Thunder and Del Sol are 827.08, 937.95 and 1045.00, respectively.

TABLE 9 Plant Height (cm) at Harvest Maturity Trial Location Loc. 1 Loc.2 Loc. 3 Loc. 4 Inferno (Inferno) 36.0 38.0 33.0 37.5 36.0 40.0 31.536.0 37.0 43.5 32.5 35.0 36.0 38.0 32.5 37.5 37.0 37.0 34.5 35.5 38.039.0 30.5 35.5 39.0 40.0 32.0 35.0 37.0 40.5 35.5 38.0 38.0 40.0 30.536.0 40.0 38.0 32.5 37.5 Green Thunder 33.0 33.5 30.0 35.0 34.0 33.032.5 41.0 36.0 34.0 31.0 39.0 35.0 34.0 32.0 39.0 34.0 35.5 36.5 41.036.0 33.0 34.0 40.0 35.0 33.5 31.0 40.0 34.0 33.0 33.5 39.0 35.0 31.536.0 41.0 36.0 35.0 28.0 41.0 Del Sol 36.0 37.0 35.5 38.0 37.0 38.0 37.040.0 35.0 39.0 33.0 39.0 36.0 37.0 35.0 38.0 37.0 37.0 35.0 39.0 35.036.0 37.0 40.0 34.0 33.0 37.5 42.0 35.0 37.5 35.5 40.0 36.0 43.0 36.541.0 37.0 34.0 35.5 41.0 Plant Height (cm) Duncan Variety Mean NGrouping Inferno 36.41 40 A Green Thunder 35.11 40 B Del Sol 37.13 40 AANOVA Source of Variation df SS MS F P-value Variety 2 83.304 41.65214.81 <.0001 Location 3 387.617 129.206 45.93 <.0001 Interaction 6268.646 44.774 15.92 <.0001 Anova shows significant differences in plantheight for varieties, locations and significant interactions betweenVariety and location. The average plant height (cm) of Inferno, GreenThunder and Del Sol are 36.41, 35.11 and 37.13, respectively.

TABLE 10 Frame Leaf Length (cm) at Harvest Maturity Trial location Loc.1 Loc. 2 Loc. 3 Loc. 4 Inferno (Inferno) 31.0 35.0 30.5 31.5 30.0 36.530.5 23.0 28.0 35.5 30.5 31.5 32.0 39.5 31.5 31.0 31.0 37.0 32.5 32.530.0 36.0 28.0 34.0 31.0 35.5 30.5 33.5 30.0 36.0 28.5 30.5 32.0 34.527.0 30.0 30.0 33.5 26.5 30.5 Green Thunder 29.0 33.5 33.0 35.0 30.030.5 33.0 35.5 32.0 31.0 26.5 38.0 30.0 30.5 31.5 35.0 32.0 30.0 33.037.0 30.0 33.0 31.0 32.5 29.0 32.0 30.0 37.0 30.0 31.5 28.0 34.0 35.034.0 30.5 36.0 30.0 30.0 26.0 32.5 Del Sol 32.0 32.0 35.0 39.0 31.0 34.035.5 35.0 31.0 34.5 35.0 36.0 30.0 31.5 33.0 40.0 35.0 34.5 33.0 38.030.0 35.5 31.0 37.0 28.0 33.5 30.0 35.0 30.0 32.5 34.0 35.0 30.0 33.030.5 39.0 29.0 33.0 32.0 39.0 Leaf Length (cm) Duncan Variety Mean NGrouping Inferno 31.70 40 B Green Thunder 31.95 40 B Del Sol 33.55 40 AANOVA Source of Variation df SS MS F P-value Variety 2 80.600 40.30010.54 <.0001 Location 3 334.917 111.639 29.20 <.0001 Interaction 6294.833 49.139 12.85 <.0001 Anova shows significant differences in frameleaf length at harvest maturity stage for varieties, locations andSignificant interactions between variety and location. The averagematured leaf length (cm) of Inferno, Green Thunder and Del Sol are31.70, 31.95 and 33.55, respectively.

TABLE 11 Leaf Width(cm) at Harvest Maturity Trial Location Loc. 1 Loc. 2Loc. 3 Loc. 4 Inferno (Inferno) 21.0 20.5 16.0 20.0 20.0 22.0 16.0 19.021.0 22.5 15.5 19.0 21.0 23.5 17.5 18.0 20.0 21.0 16.5 20.0 20.0 21.516.0 20.0 18.0 21.0 15.5 20.5 19.0 21.0 16.5 17.5 20.0 21.0 14.5 18.020.0 22.0 15.5 20.0 Green Thunder 17.0 22.5 19.5 24.0 20.0 21.5 18.023.5 21.0 21.0 16.0 25.5 18.0 19.0 18.0 23.0 20.0 20.5 14.0 26.0 18.020.5 17.0 25.5 17.0 18.5 18.0 27.0 18.0 18.5 16.5 24.0 20.0 21.0 18.023.0 18.0 23.0 17.5 24.0 Del Sol 20.0 19.0 22.5 22.0 18.0 22.0 22.0 23.019.0 20.0 20.0 21.0 20.0 19.5 20.0 25.0 22.0 20.0 19.5 25.0 18.0 22.017.5 26.0 16.0 22.0 19.5 24.0 20.0 21.5 18.0 23.0 18.0 22.0 15.0 25.016.0 21.5 18.0 23.0 Leaf Width (cm) Duncan Variety Mean N GroupingInferno 19.19 40 B Green Thunder 20.28 40 A Del Sol 20.64 40 A ANOVASource of Variation df SS MS F P-value Variety 2 45.554 22.777 11.25<.0001 Location 3 433.017 144.339 71.31 <.0001 Interaction 6 189.69631.616 15.62 <.0001 Anova shows significant differences in frame leafwidth at harvest maturity stage for varieties, locations and Significantinteractions between variety and location. The average matured leafwidth (cm) of Inferno, Green Thunder and Del Sol are 19.19, 20.28 and20.64, respectively.

TABLE 12 Leaf Index calculated by dividing the leaf length by the leafwidth Trial Location Loc. 1 Loc. 2 Loc. 3 Loc. 4 Inferno (Inferno) 1.481.71 1.91 1.58 1.50 1.66 1.91 1.21 1.33 1.58 1.97 1.66 1.52 1.68 1.801.72 1.55 1.76 1.97 1.63 1.50 1.67 1.75 1.70 1.72 1.69 1.97 1.63 1.581.71 1.73 1.74 1.60 1.64 1.86 1.67 1.50 1.52 1.71 1.53 Green Thunder1.71 1.49 1.69 1.46 1.50 1.42 1.83 1.51 1.52 1.48 1.66 1.49 1.67 1.611.75 1.52 1.60 1.46 2.36 1.42 1.67 1.61 1.82 1.27 1.71 1.73 1.67 1.371.67 1.70 1.70 1.42 1.75 1.62 1.69 1.57 1.67 1.30 1.49 1.35 Del Sol 1.601.68 1.56 1.77 1.72 1.55 1.61 1.52 1.63 1.73 1.75 1.71 1.50 1.62 1.651.60 1.59 1.73 1.69 1.52 1.67 1.61 1.77 1.42 1.75 1.52 1.54 1.46 1.501.51 1.89 1.52 1.67 1.50 2.03 1.56 1.81 1.53 1.78 1.70 Wrap Leaf IndexDuncan Variety Mean N Grouping Inferno 1.66 40 A Green Thunder 1.60 40 BDel Sol 1.64 40 AB ANOVA Source of Variation df SS MS F P-value Variety2 0.087 0.043 2.75 0.0682 Location 3 0.988 0.329 20.91 <.0001Interaction 6 0.331 0.055 3.51 0.0033 Anova shows significantdifferences in frame leaf index at harvest maturity stage for varietiesand locations, and significant interactions between variety andlocation. The average frame leaf index of Inferno, Green Thunder and DelSol are 1.66, 1.60 and 1.64, respectively.

TABLE 13 Leaf Area (cm2), calculated by multiplying the leaf length bythe leaf width Trial Location Loc. 1 Loc. 2 Loc. 3 Loc. 4 Inferno(Inferno) 651 717.5 488 630 600 803 488 437 588 798.75 472.75 598.5 672928.25 551.25 558 620 777 536.25 650 600 774 448 680 558 745.5 472.75686.75 570 756 470.25 533.75 640 724.5 391.5 540 600 737 410.75 610Green Thunder 493 753.75 643.5 840 600 655.75 594 834.25 672 651 424 969540 579.5 567 805 640 615 462 962 540 676.5 527 828.75 493 592 540 999540 582.75 462 816 700 714 549 828 540 690 455 780 Del Sol 640 608 787.5858 558 748 781 805 589 690 700 756 600 614.25 660 1000 770 690 643.5950 540 781 542.5 962 448 737 585 840 600 698.75 612 805 540 726 457.5975 464 709.5 576 897 Leaf Area Duncan Variety Mean N Grouping Inferno612.85 40 C Green Thunder 653.84 40 B Del Sol 698.61 40 A ANOVA Sourceof Variation df SS MS F P-value Variety 2 147199.132 73599.566 14.17<.0001 Location 3 1079021.552 359673.851 69.23 <.0001 Interaction 6613318.672 102219.779 19.67 <.0001 Anova shows significant differencesin leaf area (cm2) at harvest maturity stage for varieties, locationsand significant interactions between variety and location. The averageleaf area (cm2) of Inferno, Green Thunder and Del Sol are 612.85, 653.84and 698.61, respectively.

TABLE 14 Core Length (mm) at Harvest Maturity Trial Location Loc. 1 Loc.2 Loc. 3 Loc. 4 Inferno (Inferno) 70 95 65 69 75 100 46 75 80 120 46 9580 105 48 90 70 85 49 100 75 115 56 95 78 112 50 70 80 98 50 95 65 10540 92 70 90 45 100 Green Thunder 100 95 60 123 100 130 53 95 95 120 58110 105 80 50 95 110 120 53 112 105 105 55 120 95 94 60 120 95 85 60 110105 90 43 115 100 90 45 125 Del Sol 80 95 48 90 70 100 50 85 65 100 5080 80 120 50 80 82 85 50 70 78 100 45 92 70 90 45 75 78 90 45 65 80 12050 95 70 60 16 85 Core Length (mm) at Harvest Maturity Duncan VarietyMean N Grouping Inferno 78.60 40 B Green Thunder 92.03 40 A Del Sol74.48 40 B ANOVA Source of Variation df SS MS F P-value Variety 26736.650 3368.325 30.03 <.0001 Location 3 45905.267 15301.756 136.44<.0001 Interaction 6 3744.683 624.114 5.56 <.0001 Anova showssignificant differences in core length at harvest maturity stage forvarieties, locations and significant interactions between variety andlocation. The average core length (mm) of Inferno, Green Thunder and DelSol are 78.06, 92.03 and 74.48, respectively.

TABLE 15 Core Diameter (mm) at Harvest Maturity Trial Location Loc. 1Loc. 2 Loc. 3 Loc. 4 Inferno (Inferno) 35 37 35 37 35 35 30 32 36 40 2840 35 35 32 38 30 35 35 36 30 40 34 32 34 35 30 35 35 40 32 36 30 35 3037 30 35 30 37 Green Thunder 35 40 35 46 42 50 35 47 40 48 40 47 35 4535 45 40 43 35 45 35 45 40 52 40 45 37 45 30 40 35 50 40 50 40 45 40 5035 42 Del Sol 42 48 40 45 45 50 38 45 40 47 32 45 40 45 36 50 41 45 4050 35 40 35 55 34 45 40 45 35 45 40 45 44 45 38 55 40 40 38 45 CoreDiameter (mm) at Harvest Maturity Duncan Variety Mean N Grouping Inferno34.33 40 B Green Thunder 41.60 40 A Del Sol 42.58 40 A ANOVA Source ofVariation df SS MS F P-value Variety 2 1625.850 812.925 83.93 <.0001Location 3 1475.133 491.711 50.77 <.0001 Interaction 6 163.017 7.1692.81 0.0142 Anova shows significant differences in core diameter atharvest maturity stage for varieties, locations and significantinteractions between variety and location. The average core diameter(mm) of Inferno, Green Thunder and Del Sol are 34.33, 41.60 and 42.58,respectively.

TABLE 16 The Height of Mature Seed Stalk (cm) Inferno Green (Inferno)Thunder Del Sol 130 90 88 125 100 90 133 95 95 130 95 85 126 95 90 12890 95 132 100 98 130 98 100 125 95 95 128 98 98 130 95 95 125 96 90 13095 95 130 95 85 125 98 86 124 100 88 120 90 90 122 95 98 123 98 90 120100 95 Variety Mean N Duncan Grouping Inferno 126.80 20 A Green Thunder95.90 20 B Del Sol 92.30 20 C ANOVA Source of Variation df SS MS FP-value Variety 2 14386.800 7193.400 462.15 <.0001 Anova shows asignificant difference in the height of mature seed stalk, and theaverage mature seed stalk height (cm) is 126.80, 95.90 and 92.30,respectively.

TABLE 17 The spread of mature see stalk at widest point (cm) InfernoGreen (Inferno) Thunder Del Sol 35 38 38 45 45 40 44 38 43 43 40 38 4241 35 44 42 40 38 45 42 39 35 44 40 36 42 42 38 40 40 37 35 38 38 38 3638 40 40 40 42 41 42 40 42 44 45 40 45 44 38 42 45 40 35 40 41 36 44Variety Mean N Duncan Grouping Inferno 40.40 20 A Green Thunder 39.75 20A Del Sol 40.75 20 A ANOVA Source of Variation df SS MS F P-valueVariety 2 10.300 5.150 0.57 0.5683 Anova shows no significant differencein the spread of mature seed stalk at widest point.

Further Embodiments of the Invention

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign nucleic acids including additional or modified versions ofnative (endogenous) nucleic acids (optionally driven by a non-nativepromoter) in order to alter the traits of a plant in a specific manner.Any nucleic acid sequences, whether from a different species or from thesame species, which are introduced into the genome using transformationor various breeding methods, are referred to herein collectively as“transgenes.” Over the last fifteen to twenty years, several methods forproducing transgenic plants have been developed, and in particularembodiments the present invention also relates to transformed versionsof lettuce plants disclosed herein.

Genetic engineering techniques can be used (alone or in combination withbreeding methods) to introduce one or more desired added traits intoplant, for example, lettuce cultivar Inferno or progeny or lettuceplants derived thereof.

Plant transformation generally involves the construction of anexpression vector that will function in plant cells. Optionally, such avector comprises one or more nucleic acids comprising a coding sequencefor a polypeptide or an untranslated functional RNA under control of, oroperatively linked to, a regulatory element (for example, a promoter).In representative embodiments, the vector(s) may be in the form of aplasmid, and can be used alone or in combination with other plasmids, toprovide transformed lettuce plants using transformation methods asdescribed herein to incorporate transgenes into the genetic material ofthe lettuce plant.

Additional methods include, but are not limited to, expression vectorsintroduced into plant tissues using a direct nucleic acid transfermethod, such as microprojectile-mediated delivery (e.g., with abiolistic device), DNA injection, Agrobacterium-mediated transformation,electroporation, and the like. Transformed plants obtained from theplants (and parts and tissue culture thereof) of the invention areintended to be within the scope of this invention.

Expression Vectors for Plant Transformation—Selectable Markers.

Expression vectors typically include at least one nucleic acidcomprising or encoding a selectable marker, operably linked to aregulatory element (for example, a promoter) that allows transformedcells containing the marker to be either recovered by negativeselection, e.g., inhibiting growth of cells that do not contain theselectable marker, or by positive selection, e.g., screening for theproduct encoded by the selectable marker. Many commonly used selectablemarkers for plant transformation are well known in the transformationart, and include, for example, nucleic acids that code for enzymes thatmetabolically detoxify a selective chemical agent which may be anantibiotic or an herbicide, or nucleic acids that encode an alteredtarget which is insensitive to the inhibitor. Positive selection methodsare also known in the art.

One commonly used selectable marker for plant transformation is aneomycin phosphotransferase II (nptII) coding sequence, for example,isolated from transposon Tn5, which when placed under the control ofplant regulatory signals confers resistance to kanamycin. Fraley, etal., PNAS, 80:4803 (1983). Another commonly used selectable marker ishygromycin phosphotransferase, which confers resistance to theantibiotic hygromycin. Vanden Elzen, et al., Plant Mol. Biol., 5:299(1985).

Additional selectable markers of bacterial origin that confer resistanceto antibiotics include gentamycin acetyl transferase, streptomycinphosphotransferase, aminoglycoside-3′-adenyl transferase, the bleomycinresistance determinant. Hayford, et al., Plant Physiol., 86:1216 (1988);Jones, et al., Mol. Gen. Genet., 210:86 (1987); Svab, et al., Plant Mol.Biol., 14:197 (1990); Hille, et al., Plant Mol. Biol., 7:171 (1986).Other selectable markers confer resistance to herbicides such asglyphosate, glufosinate, or bromoxynil. Comai, et al., Nature,317:741-744 (1985); Gordon-Kamm, et al., Plant Cell, 2:603-618 (1990);and Stalker, et al., Science, 242:419-423 (1988).

Selectable markers for plant transformation that are not of bacterialorigin include, for example, mouse dihydrofolate reductase, plant5-enolpyruvylshikimate-3-phosphate synthase, and plant acetolactatesynthase. Eichholtz, et al., Somatic Cell Mol. Genet., 13:67 (1987);Shah, et al., Science, 233:478 (1986); and Charest, et al., Plant CellRep., 8:643 (1990).

Another class of selectable marker for plant transformation involvesscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These selectable markers areparticularly useful to quantify or visualize the spatial pattern ofexpression of a transgene in specific tissues and are frequentlyreferred to as a reporter gene because they can be fused to transgene orregulatory sequence for the investigation of nucleic acid expression.Commonly used reporters for screening presumptively transformed cellsinclude alpha-glucuronidase (GUS), alpha-galactosidase, luciferase andchloramphenicol, acetyltransferase. Jefferson, R. A., Plant Mol. Biol.,5:387 (1987); Teeri, et al., EMBO J., 8:343 (1989); Koncz, et al., PNAS,84:131 (1987); and DeBlock, et al., EMBO J., 3:1681 (1984).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissues are available. Molecular Probes,Publication 2908, IMAGENE GREEN, pp. 1-4 (1993) and Naleway, et al., J.Cell Biol., 115:151a (1991).

Green Fluorescent Protein (GFP) is also utilized as a marker for nucleicacid expression in prokaryotic and eukaryotic cells. Chalfie, et al.,Science, 263:802 (1994). GFP and mutants of GFP may be used asscreenable markers.

Expression Vectors for Plant Transformation—Promoters.

Transgenes included in expression vectors are generally driven by anucleotide sequence comprising a regulatory element (for example, apromoter). Numerous types of promoters are well known in thetransformation arts, as are other regulatory elements that can be usedalone or in combination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells.

Examples of promoters under developmental control include promoters thatpreferentially initiate transcription in certain tissues, such asleaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma.Such promoters are referred to as “tissue-preferred.” Promoters thatinitiate transcription only in certain tissue are referred to as“tissue-specific.” A “cell type” specific promoter preferentially drivesexpression in certain cell types in one or more organs, for example,vascular cells in roots or leaves. An “inducible” promoter is a promoterthat is under environmental control. Examples of environmentalconditions that may affect transcription by inducible promoters includeanaerobic conditions or the presence of light. Tissue-specific,tissue-preferred, cell type specific, and inducible promoters constitutethe class of “non-constitutive” promoters. A “constitutive” promoter isa promoter that is active under most environmental conditions.

A. Inducible Promoters:

An inducible promoter is operably linked to a nucleic acid forexpression in a plant. Optionally, the inducible promoter is operablylinked to a nucleotide sequence encoding a signal sequence which isoperably linked to a nucleic acid for expression in the plant. With aninducible promoter, the rate of transcription increases in response toan inducing agent.

Any inducible promoter can be used in the instant invention. See Ward,et al., Plant Mol. Biol., 22:361-366 (1993). Exemplary induciblepromoters include, but are not limited to, that from the ACEI systemwhich responds to copper (Melt, et al., PNAS, 90:4567-4571 (1993));promoter from the In2 gene from maize which responds tobenzenesulfonamide herbicide safeners (Hershey, et al., Mol. Gen.Genet., 227:229-237 (1991) and Gatz, et al., Mol. Gen. Genet., 243:32-38(1994)) or Tet repressor from Tn10 (Gatz, et al., Mol. Gen. Genet.,227:229-237 (1991)). A representative inducible promoter is a promoterthat responds to an inducing agent to which plants do not normallyrespond. An exemplary inducible promoter is the inducible promoter froma steroid hormone gene, the transcriptional activity of which is inducedby a glucocorticosteroid hormone. Schena, et al., PNAS, 88:0421 (1991).

B. Constitutive Promoters:

A constitutive promoter is operably linked to a nucleic acid forexpression in a plant or the constitutive promoter is operably linked toa nucleotide sequence encoding a signal sequence which is operablylinked to a nucleic acid for expression in a plant.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell, et al., Nature, 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy, et al., Plant Cell, 2:163-171 (1990));ubiquitin (Christensen, et al., Plant Mol. Biol., 12:619-632 (1989) andChristensen, et al., Plant Mol. Biol., 18:675-689 (1992)); pEMU (Last,et al., Theor. Appl. Genet., 81:581-588 (1991)); MAS (Velten, et al.,EMBO J., 3:2723-2730 (1984)) and maize H3 histone (Lepetit, et al., Mol.Gen. Genet., 231:276-285 (1992) and Atanassova, et al., Plant J., 2(3):291-300 (1992)). The ALS promoter, XbaI/NcoI fragment 5′ to theBrassica napus ALS3 structural gene (or a nucleotide sequence similarityto said XbaI/NcoI fragment), represents a particularly usefulconstitutive promoter. See PCT Application No. WO 96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters:

A tissue-specific promoter is operably linked to a nucleic acid forexpression in a plant. Optionally, the tissue-specific promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a nucleic acid for expression in a plant.Plants transformed with a nucleic acid of interest operably linked to atissue-specific promoter transcribe the nucleic acid of interestexclusively, or preferentially, in a specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai, et al., Science, 23:476-482(1983) and Sengupta-Gopalan, et al., PNAS, 82:3320-3324 (1985)); aleaf-specific and light-induced promoter such as that from cab orrubisco (Simpson, et al., EMBO J., 4(11):2723-2729 (1985) and Timko, etal., Nature, 318:579-582 (1985)); an anther-specific promoter such asthat from LAT52 (Twell, et al., Mol. Gen. Genet., 217:240-245 (1989)); apollen-specific promoter such as that from Zm13 (Guerrero, et al., Mol.Gen. Genet., 244:161-168 (1993)) or a microspore-preferred promoter suchas that from apg (Twell, et al., Sex. Plant Reprod., 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments.

Transport of polypeptides produced by transgenes to a subcellularcompartment such as the chloroplast, vacuole, peroxisome, glyoxysome,cell wall, or mitochondrion, or for secretion into the apoplast, isgenerally accomplished by means of operably linking a nucleotidesequence encoding a signal sequence to the 5′ and/or 3′ region of anucleic acid encoding the polypeptide of interest. Signal sequences atthe 5′ and/or 3′ end of the coding sequence target the polypeptide toparticular subcellular compartments.

The presence of a signal sequence can direct a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample, Becker, et al., Plant Mol. Biol., 20:49 (1992); Close, P. S.,Master's Thesis, Iowa State University (1993); Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley,” Plant Mol. Biol., 9:3-17 (1987); Lerner, et al., PlantPhysiol., 91:124-129 (1989); Fontes, et al., Plant Cell, 3:483-496(1991); Matsuoka, et al., PNAS, 88:834 (1991); Gould, et al., J. Cell.Biol., 108:1657 (1989); Creissen, et al., Plant J, 2:129 (1991);Kalderon, et al., A short amino acid sequence able to specify nuclearlocation, Cell, 39:499-509 (1984); and Steifel, et al., Expression of amaize cell wall hydroxyproline-rich glycoprotein gene in early leaf androot vascular differentiation, Plant Cell, 2:785-793 (1990).

Foreign Polypeptide Transgenes and Agronomic Transgenes.

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign polypeptide then canbe extracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem., 114:92-6(1981).

According to a representative embodiment, the transgenic plant providedfor commercial production of foreign protein is a lettuce plant of theinvention. In another embodiment, the biomass of interest is seed. Forthe relatively small number of transgenic plants that show higher levelsof expression, a genetic map can be generated, for example viaconventional RFLP, PCR, and SSR analysis, which identifies theapproximate chromosomal location of the integrated DNA molecule. Forexemplary methodologies in this regard, see Methods in Plant MolecularBiology and Biotechnology, Glick and Thompson Eds., 269:284, CRC Press,Boca Raton (1993). Map information concerning chromosomal location isuseful for proprietary protection of a subject transgenic plant. Ifunauthorized propagation is undertaken and crosses made with othergermplasm, the map of the integration region can be compared to similarmaps for suspect plants, to determine if the latter have a commonparentage with the subject plant. Map comparisons can involvehybridizations, RFLP, PCR, SSR, and sequencing, all of which areconventional techniques.

Likewise, by means of the present invention, agronomic transgenes andother desired added traits can be expressed in transformed plants (andtheir progeny, e.g., produced by breeding methods). More particularly,plants can be genetically engineered to express various phenotypes ofagronomic interest or other desired added traits. Exemplary nucleicacids of interest in this regard conferring a desired added trait(s)include, but are not limited to, those categorized below:

A. Transgenes that Confer Resistance to Pests or Disease:

1. Plant disease resistance transgenes. Plant defenses are oftenactivated by specific interaction between the product of a diseaseresistance gene (R) in the plant and the product of a correspondingavirulence (Avr) gene in the pathogen. A plant line can be transformedwith a cloned resistance transgene to engineer plants that are resistantto specific pathogen strains. See, for example, Jones, et al., Science,266:789 (1994) (cloning of the tomato Cf-9 gene for resistance toCladosporium fulvum); Martin, et al., Science, 262:1432 (1993) (tomatoPto gene for resistance to Pseudomonas syringae pv. tomato encodes aprotein kinase); and Mindrinos, et al., Cell, 78:1089 (1994)(Arabidopsis RSP2 gene for resistance to Pseudomonas syringae).

2. A Bacillus thuringiensis protein, a derivative thereof, or asynthetic polypeptide modeled thereon. See, for example, Geiser, et al.,Gene, 48:109 (1986), who disclose the cloning and nucleotide sequence ofa Bt delta-endotoxin gene. Moreover, DNA molecules encodingdelta-endotoxin transgenes can be purchased from American Type CultureCollection, Manassas, Va., for example, under ATCC Accession Nos. 40098,67136, 31995, and 31998.

3. A lectin. See, for example, the disclosure by Van Damme, et al.,Plant Mol. Biol., 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin transgenes.

4. A vitamin-binding protein such as avidin. See, e.g., PCT ApplicationNo. US 93/06487. The application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

5. An enzyme inhibitor, for example, a protease or proteinase inhibitor,or an amylase inhibitor. See, for example, Abe, et al., J. Biol. Chem.,262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor); Huub, et al., Plant Mol. Biol., 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I); and Sumitani,et al., Biosci. Biotech. Biochem., 57:1243 (1993) (nucleotide sequenceof Streptomyces nitrosporeus alpha-amylase inhibitor).

6. An insect-specific hormone or pheromone, such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock, et al., Nature, 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

7. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem., 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor) and Pratt, etal., Biochem. Biophys. Res. Comm., 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also, U.S. Pat. No. 5,266,317 toTomalski, et al., who disclose transgenes encoding insect-specific,paralytic neurotoxins.

8. An insect-specific venom produced in nature, by a snake, a wasp, etc.For example, see Pang, et al, Gene, 116:165 (1992), for disclosure ofheterologous expression in plants of a transgene coding for a scorpioninsectotoxic peptide.

9. An enzyme responsible for a hyper-accumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative,or another non-protein molecule with insecticidal activity.

10. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase, and a glucanase, whether natural or synthetic. See PCTApplication No. WO 93/02197 in the name of Scott, et al., whichdiscloses the nucleotide sequence of a callase transgene. DNA moleculeswhich contain chitinase-encoding sequences can be obtained, for example,from the ATCC under Accession Nos. 39637 and 67152. See also, Kramer, etal., Insect Biochem. Mol. Biol., 23:691 (1993), who teach the nucleotidesequence of a cDNA encoding tobacco hornworm chitinase, and Kawalleck,et al., Plant Mol. Biol., 21:673 (1993), who provide the nucleotidesequence of the parsley ubi4-2 polyubiquitin transgene.

11. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella, et al., Plant Mol. Biol., 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess,et al., Plant Physiol., 104:1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

12. A hydrophobic moment peptide. See PCT Application No. WO 95/16776(disclosure of peptide derivatives of tachyplesin which inhibit fungalplant pathogens) and PCT Application No. WO 95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance).

13. A membrane permease, a channel former, or a channel blocker. Forexample, see the disclosure of Jaynes, et al., Plant Sci., 89:43 (1993),of heterologous expression of a cecropin-beta, lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

14. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein transgene is derived,as well as by related viruses. See Beachy, et al., Ann. Rev.Phytopathol., 28:451 (1990). Coat protein-mediated resistance has beenconferred upon transformed plants against alfalfa mosaic virus, cucumbermosaic virus, tobacco streak virus, potato virus X, potato virus Y,tobacco etch virus, tobacco rattle virus, and tobacco mosaic virus. Id.

15. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. SeeTaylor, et al., Abstract #497, Seventh Intl Symposium on MolecularPlant-Microbe Interactions, Edinburgh, Scotland (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

16. A virus-specific antibody. See, for example, Tavladoraki, et al.,Nature, 366:469 (1993), who show that transgenic plants expressingrecombinant antibody transgenes are protected from virus attack.

17. A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo-alpha-1,4-D-polygalacturonasesfacilitate fungal colonization and plant nutrient released bysolubilizing plant cell wall homo-alpha-1,4-D-galacturonase. See Lamb,et al., Bio/technology, 10:1436 (1992). The cloning and characterizationof a transgene which encodes a bean endopolygalacturonase-inhibitingprotein is described by Toubart, et al., Plant J., 2:367 (1992).

18. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann, et al., Bio/technology, 10:305 (1992), have shownthat transgenic plants expressing the barley ribosome-inactivatingtransgene have an increased resistance to fungal disease.

19. A lettuce mosaic potyvirus (LMV) coat protein transgene introducedinto Lactuca sativa in order to increase its resistance to LMVinfection. See Dinant, et al., Mol. Breeding, 3:1, 75-86 (1997).

Any disease or present resistance transgenes, including thoseexemplified above, can be introduced into a lettuce plant of theinvention through a variety of means including but not limited totransformation and breeding.

B. Transgenes that Confer Resistance to an Herbicide:

Exemplary polynucleotides encoding polypeptides that confer traitsdesirable for herbicide resistance include acetolactate synthase (ALS)mutants that lead to herbicide resistance such as the S4 and/or Hramutations ((resistance to herbicides including sulfonylureas,imidazolinones, triazolopyrimidines, pyrimidinyl thiobenzoates);glyphosate resistance (e.g.,5-enol-pyrovyl-shikimate-3-phosphate-synthase (EPSPS) transgene,including but not limited to those described in U.S. Pat. Nos.4,940,935, 5,188,642, 5,633,435, 6,566,587, 7,674,598 as well as allrelated application; or the glyphosate N-acetyltransferase (GAT)transgene, described in Castle et al., Science, 2004, 304:1151-1154; andin U.S. Patent Application Publication Nos. 20070004912, 20050246798,and 20050060767)); glufosinate resistance (e.g., BAR; see e.g., U.S.Pat. No. 5,561,236); 2,4-D resistance (e.g., aryloxy alkanoatedioxygenase or AAD-1, AAD-12, or AAD-13), HPPD resistance (e.g.,Pseudomonas HPPD) and PPO resistance (e.g., fomesafen,acifluorfen-sodium, oxyfluorfen, lactofen, fluthiacet-methyl,saflufenacil, flumioxazin, flumiclorac-pentyl, carfentrazone-ethyl,sulfentrazone,); a cytochrome P450 or variant thereof that confersherbicide resistance or tolerance to, inter alia, HPPD-inhibitingherbicides, PPO-inhibiting herbicides and ALS-inhibiting herbicides(U.S. Patent Application Publication No. 20090011936; U.S. Pat. Nos.6,380,465; 6,121,512; 5,349,127; 6,649,814; and 6,300,544; and PCTInternational Publication No. WO 2007/000077); dicamba resistance (e.g.,dicamba monoxygenase), and traits desirable for processing or processproducts such as high oil (e.g., U.S. Pat. No. 6,232,529); modified oils(e.g., fatty acid desaturase transgenes (U.S. Pat. No. 5,952,544; PCTInternational Publication No. WO 94/11516)); modified starches (e.g.,ADPG pyrophosphorylases (AGPase), starch synthases (SS), starchbranching enzymes (SBE), and starch debranching enzymes (SDBE)); andpolymers or bioplastics (e.g., U.S. Pat. No. 5,602,321;beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoAreductase (Schubert et al., J. Bacteriol., 1988, 170:5837-5847)facilitate expression of polyhydroxyalkanoates (PHAs)).

In embodiments, the polynucleotide encodes a polypeptide conferringresistance to an herbicide selected from glyphosate, sulfonylurea,imidazolinone, dicamba, glufosinate, phenoxy proprionic acid,L-phosphinothricin, cyclohexone, cyclohexanedione, triazine, andbenzonitrile.

Any transgene conferring herbicide resistance, including thoseexemplified above, can be introduced into the lettuce plants of theinvention through a variety of means including, but not limited to,transformation (e.g., genetic engineering techniques) and crossing.

C. Transgenes that Confer or Contribute to a Value-Added Trait:

1. Increased iron content of the lettuce, for example, by introducinginto a plant a soybean ferritin transgene as described in Goto, et al.,Acta Horticulturae., 521, 101-109 (2000).

2. Decreased nitrate content of leaves, for example, by introducing intoa lettuce a transgene coding for a nitrate reductase. See, for example,Curtis, et al., Plant Cell Rep., 18:11, 889-896 (1999).

3. Increased sweetness of the lettuce by introducing a transgene codingfor monellin that elicits a flavor 100,000 times sweeter than sugar on amolar basis. See Penarrubia, et al., Bio/technology, 10:561-564 (1992).

4. Modified fatty acid metabolism, for example, by introducing into aplant an antisense sequence directed against stearyl-ACP desaturase toincrease stearic acid content of the plant. See Knultzon, et al., PNAS,89:2625 (1992).

5. Modified carbohydrate composition effected, for example, byintroducing into plants a transgene coding for an enzyme that alters thebranching pattern of starch. See Shiroza, et al., J. Bacteria, 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase transgene); Steinmetz, et al., Mol. Gen. Genet.,20:220 (1985) (nucleotide sequence of Bacillus subtilis levansucrasetransgene); Pen, et al., Bio/technology, 10:292 (1992) (production oftransgenic plants that express Bacillus lichenifonnis alpha-amylase);Elliot, et al., Plant Mol. Biol., 21:515 (1993) (nucleotide sequences oftomato invertase transgenes); Sogaard, et al., J. Biol. Chem., 268:22480(1993) (site-directed mutagenesis of barley alpha-amylase transgene);and Fisher, et al., Plant Physiol., 102:1045 (1993) (maize endospermstarch branching enzyme II).

Any transgene that confers or contributes a value-added trait, includingthose exemplified above, can be introduced into the lettuce plants ofthe invention through a variety of means including, but not limited to,transformation (e.g., genetic engineering techniques) and crossing.

D. Transgenes that Control Male-Sterility:

1. Introduction of a deacetylase transgene under the control of atapetum-specific promoter and with the application of the chemicalN—Ac-PPT. See, e.g., International Publication WO 01/29237.

2. Introduction of various stamen-specific promoters. See, e.g.,International Publications WO 92/13956 and WO 92/13957.

3. Introduction of the barnase and the barstar transgenes. See, e.g.,Paul, et al., Plant Mol. Biol., 19:611-622 (1992).

Any transgene that controls male sterility, including those exemplifiedabove, can be introduced into the lettuce plants of the inventionthrough a variety of means including, but not limited to, transformation(e.g., genetic engineering techniques) and crossing.

Methods for Plant Transformation.

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki, et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glickand Thompson Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993). Inaddition, expression vectors and in vitro culture methods for plant cellor tissue transformation and regeneration of plants are available. See,for example, Gruber, et al., “Vectors for Plant Transformation” inMethods in Plant Molecular Biology and Biotechnology, Glick and ThompsonEds., CRC Press, Inc., Boca Raton, pp. 89-119 (1993).

A. Agrobacterium-Mediated Transformation.

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch, et al., Science, 227:1229 (1985); Curtis, et al., Journal ofExperimental Botany, 45:279, 1441-1449 (1994); Torres, et al., PlantCell Tissue and Organ Culture, 34:3, 279-285 (1993); and Dinant, et al.,Molecular Breeding, 3:1, 75-86 (1997). A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria which genetically transform plantcells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant Sci., 10:1(1991). Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated transgene transfer are provided by Gruber, etal., supra, Miki, et al., supra, and Moloney, et al., Plant Cell Rep.,8:238 (1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

B. Direct Transgene Transfer.

Several methods of plant transformation collectively referred to asdirect transgene transfer have been developed as an alternative toAgrobacterium-mediated transformation. A generally applicable method ofplant transformation is microprojectile-mediated transformation whereinDNA is carried on the surface of microprojectiles measuring 1 micron to4 micron. The expression vector is introduced into plant tissues with abiolistic device that accelerates the microprojectiles to speeds of 300m/s to 600 m/s which is sufficient to penetrate plant cell walls andmembranes. Russell, D. R., et al., Plant Cell Rep., 12 (3, January),165-169 (1993); Aragao, F. J. L., et al., Plant Mol. Biol., 20 (2,October), 357-359 (1992); Aragao, F. J. L., et al., Plant Cell Rep., 12(9, July), 483-490 (1993); Aragao, Theor. Appl. Genet., 93:142-150(1996); Kim, J., Minamikawa, T., Plant Sci., 117:131-138 (1996);Sanford, et al., Part. Sci. Technol., 5:27 (1987); Sanford, J. C.,Trends Biotech., 6:299 (1988); Klein, et al., Bio/technology, 6:559-563(1988); Sanford, J. C., Physiol. Plant, 7:206 (1990); Klein, et al.,Bio/technology, 10:268 (1992).

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang, et al., Bio/technology, 9:996 (1991).Alternatively, liposome and spheroplast fusion have been used tointroduce expression vectors into plants. Deshayes, et al., EMBO J.,4:2731 (1985) and Christou, et al., PNAS, 84:3962 (1987). Direct uptakeof DNA into protoplasts using CaCl.sub.2 precipitation, polyvinylalcohol, or poly-L-ornithine has also been reported. Hain, et al., Mol.Gen. Genet., 199:161 (1985) and Draper, et al., Plant Cell Physiol.,23:451 (1982). Electroporation of protoplasts and whole cells andtissues have also been described. Saker, M., Kuhne, T., BiologiaPlantarum, 40(4):507-514 (1997/98); Donn, et al., In Abstracts of VIIthInternational Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p.53 (1990); D'Halluin, et al., Plant Cell, 4:1495-1505 (1992); andSpencer, et al., Plant Mol. Biol., 24:51-61 (1994). See also Chupean, etal., Bio/technology, 7:5, 503-508 (1989).

Following transformation of plant target tissues, expression of theabove-described selectable marker transgenes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic lettuce line. The transgenic lettuce line couldthen be crossed with another (non-transformed or transformed) line inorder to produce a new transgenic lettuce line. Alternatively, a genetictrait that has been engineered into a particular plant cultivar usingthe foregoing transformation techniques could be introduced into anotherline using traditional breeding (e.g., backcrossing) techniques that arewell known in the plant breeding arts. For example, a backcrossingapproach could be used to move an engineered trait from a public,non-elite inbred line into an elite inbred line, or from an inbred linecontaining a foreign transgene in its genome into an inbred line orlines which do not contain that transgene. As used herein, “crossing”can refer to a simple X by Y cross, or the process of backcrossing,depending on the context.

Gene Conversions.

When the term “lettuce plant” is used in the context of the presentinvention, this term also includes any gene conversions of that plant orvariety. The term “gene converted plant” as used herein refers to thoselettuce plants that are developed, for example, by backcrossing, geneticengineering and/or mutation, wherein essentially all of the desiredmorphological and physiological characteristics of a variety (e.g., darkgreen color and/or slow bolting and/or resistance to TBSV and/or downymildew and/or tolerance to tip burn, heat stress and/or cold stress) arerecovered in addition to the one or more genes transferred into thevariety. To illustrate, backcrossing methods can be used with thepresent invention to improve or introduce a characteristic into thevariety. The term “backcrossing” as used herein refers to the repeatedcrossing of a hybrid progeny back to the recurrent parent, e.g.,backcrossing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more times to the recurrentparent. The parental plant that contributes the gene for the desiredcharacteristic is termed the “nonrecurrent” or “donor parent.” Thisterminology refers to the fact that the nonrecurrent parent is generallyused one time in the breeding e.g., backcross) protocol and thereforedoes not recur. The gene that is transferred can be a native gene, amutated native gene or a transgene introduced by genetic engineeringtechniques into the plant (or ancestor thereof). The parental plant intowhich the gene(s) from the nonrecurrent parent are transferred is knownas the “recurrent” parent as it is used for multiple rounds in thebackcrossing protocol. Poehlman & Sleper (1994) and Fehr (1993). In atypical backcross protocol, the original variety of interest (recurrentparent) is crossed to a second variety (nonrecurrent parent) thatcarries the gene(s) of interest to be transferred. The resulting progenyfrom this cross are then crossed again to the recurrent parent and theprocess is repeated until a plant is obtained wherein essentially all ofthe desired morphological and physiological characteristics of therecurrent parent are recovered in the converted plant in addition to thetransferred gene(s) and associated trait(s) from the nonrecurrentparent.

Many gene traits have been identified that are not regularly selected inthe development of a new line but that can be improved by backcrossingtechniques. Gene traits may or may not be transgenic. Examples of thesetraits include, but are not limited to, male sterility, modified fattyacid metabolism, modified carbohydrate metabolism, herbicide resistance,pest or disease resistance (e.g., resistance to bacterial, fungal, orviral disease), insect resistance, enhanced nutritional quality,increased sweetness, increased flavor, improved ripening control,improved salt tolerance, industrial usage, yield stability, and yieldenhancement. These genes are generally inherited through the nucleus.

Tissue Culture.

Further reproduction of lettuce plants variety can occur by tissueculture and regeneration. Tissue culture of various tissues of lettuceand regeneration of plants therefrom is well known and widely published.For example, reference may be had to Teng, et al., HortScience, 27:9,1030-1032 (1992); Teng, et al., HortScience, 28:6, 669-1671 (1993);Zhang, et al., Journal of Genetics and Breeding, 46:3, 287-290 (1992);Webb, et al., Plant Cell Tissue and Organ Culture, 38:1, 77-79 (1994);Curtis, et al., Journal of Experimental Botany, 45:279, 1441-1449(1994); Nagata, et al., Journal for the American Society forHorticultural Science, 125:6, 669-672 (2000); and Ibrahim, et al., PlantCell Tissue and Organ Culture, 28(2), 139-145 (1992). It is clear fromthe literature that the state of the art is such that these methods ofobtaining plants are routinely used and have a very high rate ofsuccess. Thus, another aspect of this invention is to provide cellswhich upon growth and differentiation produce lettuce plants havingdesired characteristics of lettuce cultivar Inferno (e.g., dark greencolor and/or slow bolting and/or resistance to TBSV and/or downy mildewand/or tolerance to tip burn, heat stress and/or cold stress).Optionally, lettuce plants can be regenerated from the tissue culture ofthe invention comprising all or essentially all of the physiological andmorphological characteristics of lettuce cultivar Inferno.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, meristematic cells, andplant cells that can generate tissue culture that are intact in plantsor parts of plants, such as leaves, pollen, embryos, roots, root tips,anthers, pistils, flowers, seeds, petioles, suckers, and the like. Meansfor preparing and maintaining plant tissue culture are well known in theart. By way of example, a tissue culture comprising organs has been usedto produce regenerated plants. U.S. Pat. Nos. 5,959,185, 5,973,234, and5,977,445 describe certain techniques.

Additional Breeding Methods.

This invention is also directed to methods for producing a lettuce plantby crossing a first parent lettuce plant with a second parent lettuceplant wherein the first or second parent lettuce plant is a plant oflettuce cultivar Inferno. Further, both first and second parent lettucecan come from lettuce cultivar Inferno. Thus, any of the followingexemplary methods using lettuce cultivar Inferno are part of thisinvention: selfing, backcrosses, hybrid production, crosses topopulations, double haploid production, and the like. All plantsproduced using lettuce cultivar Inferno as at least one parent arewithin the scope of this invention, including those developed fromlettuce plants derived from lettuce cultivar Inferno. Advantageously,lettuce cultivar Inferno can be used in crosses with other, different,lettuce plants to produce the first generation (F₁) lettuce hybrid seedsand plants with desirable characteristics. The lettuce plants of theinvention can also be used for transformation where exogenous transgenesare introduced and expressed by the plants of the invention. Geneticvariants created either through traditional breeding methods or throughtransformation of the cultivars of the invention by any of a number ofprotocols known to those of skill in the art are intended to be withinthe scope of this invention.

The following describes exemplary breeding methods that may be used withlettuce cultivar Inferno in the development of further lettuce plants.One such embodiment is a method for developing lettuce cultivar Infernoprogeny lettuce plants in a lettuce plant breeding program comprising:obtaining a plant, or a part thereof, of lettuce cultivar Inferno,utilizing said plant or plant part as a source of breeding material, andselecting a lettuce cultivar Inferno progeny plant with molecularmarkers in common with lettuce cultivar Inferno and/or with some, all oressentially all morphological and/or physiological characteristics oflettuce cultivar Inferno (e.g., dark green color and/or slow boltingand/or resistance to TBSV and/or downy mildew and/or tolerance to tipburn, heat stress and/or cold stress; see also Table 1). Inrepresentative embodiments, the progeny plant has at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more of the morphological and physiologicalcharacteristics of lettuce cultivar Inferno (e.g., as described in Table1), or even all of the morphological and physiological characteristicsof lettuce cultivar Inferno so that said progeny lettuce plant is notsignificantly different for said traits than lettuce cultivar Inferno,as determined at the 5% significance level when grown in the sameenvironmental conditions; optionally, with the presence of one or moredesired additional traits (e.g., male sterility, disease resistance,pest or insect resistance, herbicide resistance, and the like). Breedingsteps that may be used in the breeding program include pedigreebreeding, backcrossing, mutation breeding and/or recurrent selection. Inconjunction with these steps, techniques such as RFLP-enhancedselection, genetic marker enhanced selection (for example, SSR markers)and/or and the making of double haploids may be utilized.

Another representative method involves producing a population of lettucecultivar Inferno progeny plants, comprising crossing lettuce cultivarInferno with another lettuce plant, thereby producing a population oflettuce plants that, on average, derives at least 6.25%, 12.5%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% of its alleles from lettuce cultivar Inferno. Oneembodiment of this invention is the lettuce plant produced by thismethod and that has obtained at least 6.25%, 12.5%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%of its alleles from lettuce cultivar Inferno. A plant of this populationmay be selected and repeatedly selfed or sibbed with a lettuce plantresulting from these successive filial generations. Another approach isto make double haploid plants to achieve homozygosity.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr and Walt, Principles of CultivarDevelopment, pp. 261-286 (1987). Thus the invention includes lettucecultivar Inferno progeny lettuce plants characterized by dark greencolor and/or slow bolting and/or resistance to TBSV and/or downy mildewand/or tolerance to tip burn, heat stress and/or cold stress. Inembodiments, the invention encompasses progeny plants having acombination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of thecharacteristics as described herein for lettuce cultivar Inferno, sothat said progeny lettuce plant is not significantly different for saidtraits than lettuce cultivar Inferno, as determined at the 5%significance level when grown in the same environmental conditions.Using techniques described herein and those known in the art, molecularmarkers may be used to identify said progeny plant as progeny of lettucecultivar Inferno. Mean trait values may be used to determine whethertrait differences are significant, and optionally the traits aremeasured on plants grown under the same environmental conditions.

Progeny of lettuce cultivar Inferno may also be characterized throughtheir filial relationship with lettuce cultivar Inferno, as for example,being within a certain number of breeding crosses of lettuce cultivarInferno. A breeding cross is a cross made to introduce new genetics intothe progeny, and is distinguished from a cross, such as a self or a sibcross or a backcross to Inferno as a recurrent parent, made to selectamong existing genetic alleles. The lower the number of breeding crossesin the pedigree, the closer the relationship between lettuce cultivarInferno and its progeny. For example, progeny produced by the methodsdescribed herein may be within 1, 2, 3, 4, 5 or more breeding crosses oflettuce cultivar Inferno.

In representative embodiments, a lettuce plant derived from lettucecultivar Inferno comprises cells comprising at least one set ofchromosomes derived from lettuce cultivar Inferno. In embodiments, thelettuce plant or population of lettuce plants derived from lettucecultivar Inferno comprises, on average, at least 6.25%, 12.5%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%,98% or 99% of its alleles from lettuce cultivar Inferno. In embodiments,the lettuce plant derived from lettuce cultivar Inferno is one, two,three, four, five or more breeding crosses removed from lettuce cultivarInferno.

In representative embodiments, a plant derived from lettuce cultivarInferno is a double haploid plant, a hybrid plant or an inbred plant.

In embodiments, a hybrid or derived plant from lettuce cultivar Infernocomprises a desired added trait. In representative embodiments, alettuce plant derived from lettuce cultivar Inferno comprises all of themorphological and physiological characteristics of lettuce cultivarInferno (e.g., as described in Table 1). In embodiments, the lettuceplant derived from lettuce cultivar Inferno comprises essentially all ofthe morphological and physiological characteristics of lettuce cultivarInferno (e.g., as described in Table 1), with the addition of a desiredadded trait.

Those skilled in the art will appreciate that any of the traitsdescribed above with respect to plant transformation methods can beintroduced into a plant of the invention (e.g., lettuce cultivar Infernoand hybrid lettuce plants and other lettuce plants derived therefrom)using breeding techniques.

DEPOSIT INFORMATION

Applicants have made a deposit of at least 2500 seeds of lettucecultivar Inferno with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va., 20110-2209 U.S.A. under ATCCDeposit No PTA-120418. This deposit of lettuce variety Inferno will bemaintained in the ATCC depository, which is a public depository, for aperiod of 30 years, or 5 years after the most recent request, or for theeffective life of the patent, whichever is longer, and will be replacedif any of the deposited seed becomes nonviable during that period.Additionally, Applicants have satisfied all the requirements of 37C.F.R. §§1.801-1.809, including providing an indication of the viabilityof the samples. During the pendency of this application, access to thedeposited material will be afforded to the Commissioner on request. Allrestrictions on the availability of the deposited material from the ATCCto the public will be irrevocably removed upon granting of the patent.Applicants impose no restrictions on the availability of the depositedmaterial from the ATCC; however, Applicants have no authority to waiveany restrictions imposed by law on the transfer of biological materialor its transportation in commerce. Applicants do not waive anyinfringement of its rights granted under this patent or under the PlantVariety Protection Act (7 USC §2321 et seq.).

Access to this deposit will be available during the pendency of thisapplication to persons determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. Upon allowance of any claims in this application, all restrictionson the availability to the public of the variety will be irrevocablyremoved by affording access to a deposit of at least 2500 seeds of thesame variety with the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va., 20110-2209 U.S.A.

The foregoing invention has been described in detail by way ofillustration and example for purposes of clarity and understanding.However, it will be apparent that certain changes and modifications suchas single gene modifications and mutations, somaclonal variants, variantindividuals selected from large populations of the plants of the instantinbred and the like may be practiced within the scope of the invention.

What is claimed is:
 1. A seed of lettuce cultivar Inferno, arepresentative sample of seed having been deposited under ATCC AccessionNo. PTA-120418.
 2. A plant of lettuce cultivar Inferno, a representativesample of seed having been deposited under ATCC Accession No.PTA-120418.
 3. A lettuce plant, or a part thereof, having all thephysiological and morphological characteristics of the lettuce plant ofclaim
 2. 4. A plant comprising at least one set of chromosomes derivedfrom the plant of claim
 2. 5. A plant comprising, on average, at least50% of the alleles of the plant of claim
 2. 6. A part of the lettuceplant of claim
 2. 7. Pollen of the plant of claim
 2. 8. An ovule of theplant of claim
 2. 9. A tissue culture of regenerable cells of the plantof claim
 2. 10. The tissue culture of claim 9, wherein the cells are:(a) embryos, meristem, leaves, pollen, cotyledons, hypocotyls, roots,root tips, anthers, flowers, pistils, ovules, seed, shoots, stems,stalks, petioles, pith and/or capsules; or (b) callus or protoplastsderived from the cells of (a).
 11. A lettuce plant regenerated from thetissue culture of claim 9, wherein the regenerated lettuce plantexpresses all of the physiological and morphological characteristics oflettuce cultivar Inferno, representative seed of said lettuce cultivarhaving been deposited under ATCC Accession No. PTA-120418.
 12. Aprocessed product from the plant of claim 2, wherein the processedproduct comprises cut, sliced, ground, pureed, dried, canned, jarred,washed, packaged, frozen and/or heated leaves.
 13. A method of producinglettuce seed, the method comprising crossing the plant of claim 2 withitself or a second lettuce plant and allowing seed to form.
 14. A seedproduced by the method of claim
 13. 15. A plant produced by growing theseed of claim
 14. 16. A method for producing a seed of a lettuce plantderived from the plant of claim 2, the method comprising: (a) crossing aplant of lettuce cultivar Inferno, a representative sample of seed oflettuce cultivar Inferno having been deposited under ATCC Accession No.PTA-120418, with a second lettuce plant; and (b) allowing seed of alettuce plant derived from lettuce cultivar Inferno to form; (c) growinga plant from the seed derived from lettuce cultivar Inferno of step (b);(d) selfing the plant grown from the lettuce seed derived from lettucecultivar Inferno or crossing it to a second lettuce plant to formadditional lettuce seed derived from lettuce cultivar Inferno; (e)repeating (c) and (d) for 0 or more times to generate further derivedlettuce seed.
 17. A seed produced by the method of claim 16, wherein theseed comprises, on average, at least 50% of the alleles of lettucecultivar Inferno, representative seed of said lettuce cultivar havingbeen deposited under ATCC Accession No. PTA-120418.
 18. A plant producedby growing the seed of claim
 17. 19. A method of vegetativelypropagating the plant of claim 2, the method comprising: (a) collectingtissue capable of being propagated from a plant of lettuce cultivarInferno, a representative sample of seed having been deposited underATCC Accession No. PTA-120418; (b) cultivating the tissue to obtainproliferated shoots; and (c) rooting the proliferated shoots to obtainrooted plantlets.
 20. The method of claim 19, further comprising growingplants from the rooted plantlets.
 21. A lettuce plant obtained by themethod of claim 20, wherein the lettuce slant expresses all of thephysiological and morphological characteristics of lettuce cultivarInferno, representative seed of said lettuce cultivar having beendeposited under ATCC Accession No. PTA-120418.
 22. A method ofintroducing a desired added trait into lettuce cultivar Inferno, themethod comprising: (a) crossing the plant of claim 2 with a lettuceplant that comprises a desired added trait to produce F1 progeny; (b)selecting an F1 progeny that comprises the desired added trait; (c)crossing the selected F1 progeny with lettuce cultivar Inferno toproduce backcross progeny; (d) selecting backcross progeny comprisingthe desired added trait and essentially all of the physiological andmorphological characteristics of the lettuce cultivar Inferno; and (e)repeating steps (c) and (d) one or more times in succession to produce aplant derived from lettuce cultivar Inferno comprising a desired addedtrait and essentially all of the physiological and morphologicalcharacteristics of the lettuce cultivar Inferno.
 23. The method of claim22, wherein the desired added trait is male sterility, pest resistance,insect resistance, disease resistance, herbicide resistance, or anycombination thereof.
 24. A lettuce plant produced by the method of claim22, wherein the lettuce plant has the desired added trait.
 25. Seed ofthe plant of claim
 24. 26. A method of producing a plant of lettucecultivar Inferno comprising a desired added trait, the method comprisingintroducing a transgene conferring the desired trait into the plant ofclaim
 2. 27. A lettuce plant produced by the method of claim 26, whereinthe lettuce plant has the desired added trait.
 28. Seed of the plant ofclaim
 27. 29. A method of producing a lettuce leaf, the methodcomprising: (a) growing the lettuce plant according to claim 2 toproduce a lettuce leaf; and (b) harvesting the lettuce leaf.
 30. Amethod of producing a lettuce leaf, the method comprising: (a) growingthe lettuce plant according to claim 24 to produce a lettuce leaf; and(b) harvesting the lettuce leaf.